Process for isolation, purification, and recrystallization of lutein from saponified marigold oleoresin and uses thereof

ABSTRACT

A method of isolating, purifying and recrystallizing substantially pure lutein, preferably from saponified marigold oleoresin in its pure free form, apart from chemical impurities and other carotenoids. Lutein may be used as an analytical standard or in cancer prevention trials and as a safe and effective color additive for human food.

NOTICE

More than one reissue application has been filed for the reissue of U.S.Pat. No. 5,382,714. The reissue applications are the presentcontinuation reissue patent application, application Ser. No. 11/150,599filed Jun. 10, 2005 (reissue of U.S. Pat. No. 5,382,714 ), applicationSer. No. 11/330,423 (divisional reissue of U.S. Pat. No. 5,382,714 ) andapplication Ser. No. 11/782,117 (continuation reissue application ofU.S. Pat. No. 5,382,714 ).

BACKGROUND OF THE INVENTION

Lutein is a naturally occurring carotenoid that has no vitamin Aactivity. There are three asymmetric centers in lutein at C-3, C-3′, andC-6′ positions. The absolute configuration of lutein in foods (fruitsand vegetables) and human serum/plasma is known to be 3R,3′R,6′R. Thisconfigurational isomer of lutein which is the most abundant form oflutein is also known as lutein A. However in human serum/plasma a smallamount of another configurational isomer of lutein, namely(3R,3′S,6′R)-lutein also known as 3′-epilutein or lutein B has beenshown to be present. The chemical structures of lutein and 3′-epiluteinare shown below. Other known configurational isomers of lutein have onlybeen isolated from the integument of marine fishes.

The terms all-E- and Z-isomers are new terminology used for lutein andother carotenoids, and refer to old terminology of all-trans and cisisomers of these compounds, respectively. Unless specified lutein refersto all -E (all-trans) isomer.

Numerous epidemiological studies in various populations have shown thatthe consumption of substantial amounts of fruits and vegetables reducethe risk of cancer. One hypothesis is that the carotenoids in thesefoods act as antioxidants through a free radical mechanism by quenchingsinglet oxygen and other oxidizing species resulting in the terminationof free radical chain reactions and in the prevention of cellularoxidative damage. The correlation between dietary carotenoids andcarotenoids found in human serum or plasmid indicate that only selectedgroups of carotenoids make their way into the human bloodstream to exerttheir effect. To date, 19 carotenoids have been identified to be presentin human blood. With the exception of β-carotene, none of thesecarotenoids have been studied for their anti-cancer activity.

There has been recent evidence to suggest that lutein, one of the mostabundant carotenoids in the diet and in human blood possesses strongantioxidant capabilities and may be useful in reduction of the incidenceof cancer. The allylic hydroxyl group at the C-3′ position of the E-endgroup of this compound can readily undergo oxidation as a result ofactivation by the neighboring double bond. The non-allylic hydroxylgroup at the C-3 position of the β-end group can also activate the C-4carbon allylic to the double bond making this carbon highly susceptibleto direct oxidation. These oxidation reactions result in the formationof four metabolites of lutein, which have been isolated andcharacterized from extracts of human serum or plasma. Further evidencefor an in vivo oxidation of lutein has also been obtained in humanfeeding studies. To date, pure lutein suitable for human use has notbeen commercially available for use as a chemopreventive agent inclinical trials. Pure lutein, free from chemical contaminants andsuitable for human consumption, is needed to design and conduct properhuman intervention studies.

Lutein is one of the major constituents of green vegetables and fruitssuch as broccoli, green beans, green peas, lima beans, Brussels sprouts,cabbage, kale, spinach, lettuce, kiwi, and honeydew. The lutein in thesegreen vegetables and fruits exists naturally in the free non-esterifiedform and co-exists with other carotenoids.

However, the isolation and purification of free form, pure lutein inlarge quantities from green vegetables is not economical. Many expensiveand time-consuming purification steps are required to remove and purifylutein from the large quantities of chlorophylls, β-carotene, andcarotenoids epoxides that are also present in green vegetables.

Lutein is also abundant in a number of yellow/orange fruits andvegetables such as mango, papaya, peaches, prunes, acorn squash, wintersquash, and oranges. Lutein in these yellow/orange fruits and vegetablesexists in the esterified form with fatty acids such as myristic, lauric,and palmitic acids. Upon ingestion of these foods, lutein esters undergohydrolysis to regenerate free lutein which is then absorbed andmetabolized by the body. However, these yellow/orange fruits andvegetables also contain high concentrations of a number of othercarotenoids which make the isolation and purification of lutein costlyand time-consuming.

Marigold flower petals are an excellent source of lutein because theycontain high levels of lutein and no significant levels of othercarotenoids. Extracts of marigold flowers are commercially available butconsist of lutein that is esterified with fatty acids such as lauric,myristic, and palmitic acids. Lutein in its natural form as it exists inmarigold flowers does not exist as free lutein. Upon saponification ofthe marigold extract, the lutein fatty acid esters are converted tolutein. However, the resulting lutein is still contaminated with anumber of chemical impurities. To date, no method has been described toisolate and purify the free form of lutein from these chemicalimpurities.

The saponified extracts of marigold flower petals are commerciallyavailable and currently used in chicken feed to enhance the yellow colorof egg yolk and the skin of chickens. However, the extract is notacceptable as a direct color additive for human foods because of thepresence of impurities. The availability of substantially pure luteinsuitable for human use and the evidence that significant levels oflutein derivatives are normally found in human blood would also makelutein an attractive color additive.

While chemical processes for synthesis of lutein from commerciallyavailable starting materials are known, such processes are extremelytime-consuming, involve multiple steps, and to date have simply notprovided an economical process for production of lutein. It thereforeappears that the most likely economic route to substantially pure luteinis through a process that extracts, isolates, and purifies lutein frommarigold flowers. Such substantially pure lutein, if economicallyavailable could be used in cancer prevention studies, as well as anattractive, naturally-occurring, non-harmful color additive in humanfoods.

It is therefore evident that there is a need for a carotenoidcomposition of substantially pure lutein. The primary objective of thepresent invention is the fulfillment of this need.

Another objective of the present invention is to provide a method forisolating, purifying and recrystallization lutein in high purity from asaponified marigold extract.

Another objective of the present invention is to provide a method forisolating, purifying and recrystallizing lutein in high purity which iseconomical to perform with a minimum of processing steps.

Another objective of the present invention is to provide purified luteinin crystalline form such that it is acceptable for human consumption anduse in cancer prevention trials and treatments without causing toxicside effects due to residual impurities.

Yet a further objective of the present invention is to provide purifiedlutein in crystalline form such that it is acceptable as a coloradditive in human foods.

The method and means of accomplishing each of the above objectives aswell as others will become apparent from the detailed description of theinvention which follows hereinafter.

SUMMARY OF THE INVENTION

The present invention relates to an efficient method of isolating,purifying, and recrystallizing substantially pure lutein preferably froma saponified marigold oleoresin. Lutein purified according to thismethod is free from a number of chemical impurities that are knownconstituents of flowers and plants. Lutein, in pure form may be used asan analytical standard and in cancer prevention trials, and as a safeand effective color additive in human food. The method involvespurifying, preferably, a saponified marigold extract through use of aseries of filtrations and water/alcohol washes to obtain crude luteincrystals. The crystals are dissolved in a halogenated organic solvent inwhich lutein is strongly soluble and then a second organic solvent inwhich lutein is only partially soluble. Upon cooling of the mixtureLutein is recrystallized in a high purity and then filtered and driedunder vacuum.

DETAILED DESCRIPTION OF THE INVENTION

A saponified marigold oleoresin commercially available under thetrademark “Kemin Yellow Oil” from Kemin Industries, Inc., Des Moines,Iowa, is a preferred starting material. While other extracts of marigoldflowers may be used, this particular one from marigold petals is mostpreferred because it is substantially free from other carotenoids.Generally, the concentration of other carotenoids in the startingmaterial should be 10% or less. The starting material, preferably themarigold petal extract available as “Kemin Yellow Oil” is homogenizedwith a mixture of distilled water and ethanol, preferably at 5° C.-10°C. for about 30 minutes to remove water soluble impurities such asanthocyanins (typical red pigments found in extracts from flowers). Theratio of distilled water to alcohol should be from 3 to 1, preferably2.3 to 1.

The saponified marigold oleoresin is derived from an organic solventextraction of dried marigold flowers (Tagetes erecta) and then treatedwith an alkaline solution resulting in lutein free from fatty acids suchas palmitic, myristic and lauric acids. At the preferred ratio ofwater/ethanol of 2.3:1 (within the range of from 3 to 1) and at the coldtemperature employed, lutein precipitates out from saponified marigoldoleoresin as orange crystals which are collected upon filtration. Otherratios of water/alcohol not within the range specified resulted in lowerrecoveries of lutein. The filtrate contains potassium hydroxide (fromthe previous saponification step of marigold extract employed by themanufacturer of the starting material), anthocyanins, and water solubleflavonoids.

Instead of ethanol, other alcohols such as methanol or isopropyl alcoholmay also be employed. However, since the purified lutein is required notto contain even traces of any toxic chemicals, the use of methanolinstead of food grade ethanol in this purification process is notnormally recommended.

the cold temperatures of 5° C.-10° C. result in the best recoveries forlutein, since this compound is not very well soluble in cold alcoholsolutions. At higher temperatures, however, solubility of lutein inalcohol is increased which of course results in lower yields for thiscompound. Temperatures within the above range provide economical yields.

The retained orange precipitate of lutein is then washed with distilledwater until the filtrate is almost colorless and the pH is near neutral.This ensures the complete removal of anthocyanins (red pigment) and thepotassium hydroxide from the precipitated lutein.

The precipitate is then washed sequentially with cold (0° C.-5° C.)alcohol, preferably ethanol and then preferably with hexane. Thesesequential washes result in the removal of the plant sterols and thefinal wash with hexane also results in removal of alcohol from lutein.As a result the crystals of this compound are dried at a faster rate dueto the evaporation of the volatile hexane. The hexane wash also removesmost of α- and β-cryptoxanthin, and particularly β-carotene which arepresent in very low concentrations in the saponified marigold extracts.

Instead of hexane, other straight chain hydrocarbons such as pentane,heptane, or petroleum ether (b.p. =30° C.-60° C.) can also beeffectively used since lutein has very low solubility in all of theseorganic solvents. The resulting orange crystals of lutein obtained atthis point are normally about 70% pure as determinedspectrophotometrically. While 70% pure lutein may be acceptable for usein animal feed, further purification of this compound may be employed toproduce lutein with purity greater than 97% 90% for human consumption.

Final purification of lutein is accomplished by recrystallization from abinary solvent system, preferably a 1:1 mixture of dichloromethane andhexane. This binary solvent system has never been used in thepurification of lutein. The choice of dichloromethane is because of theexcellent solubility of lutein in this solvent. Instead ofdichloromethane other halogenated solvents such as chloroform or1,2-dichloroethane can also be used; and instead of hexane one can alsouse other straight chain hydrocarbons such as pentane, heptane, andpetroleum ether (b.p. 30°-60° C.) Therefore, partially purified lutein(70% pure) is dissolved in a minimum amount of dichloromethanepreferably containing 1% triethylamine and hexane is added until thesolution becomes cloudy. The 1% addition of triethylamine todichloromethane neutralizes the trace amount of the acids that arenormally present in halogenated solvents. These residual acids couldresult in conversion of lutein to another carotenoid known as2′,3′-anhydrolutein. Thus, the 1% addition of triethylamine is preferredto prevent lutein from undergoing side reactions and to maximize theyield of pure lutein.

The above cloudy solution is kept within the range of from −20° C. to−10° C. to commence recrystallization. At this range of temperature, therecrystallization is most efficient and it is usually completed within 2to 3 hours. Storage of the solution at 0° C. prolongs therecrystallization process which may take as long as 24 hours and therecovery of pure lutein is not optimum. The crystals of lutein are thenfiltered off and washed, preferably with cold (0° C.) hexane. The purecrystalline lutein is dried in vacuo, i.e., at 50° C. for three days.

The purity of the resulting lutein is usually greater than 90%, mostoften greater than 97% as determined by UV/visible spectrophotometry.Based on quantitative HPLC analysis, the purified lutein consists of94.79% lutein, 3.03% of its geometrical isomers, and a total of 2.18% of2′, 3′-anhydrolutein, zeaxanthin, α-cryptoxanthin, and β-cryptoxanthin.The presence of the low levels of these carotenoids is not of anyconcern since these carotenoids are of dietary origin and are foundroutinely at much higher concentrations relative to that of lutein inhuman serum or plasma. NMR analysis showed no residual solvents (i.e.,dichloromethane, hexane, ethanol, triethylamine) or other non-carotenoidimpurities. The lutein purified according to this method exists insubstantially purer form in comparison with lutein found in the matrixof any naturally occurring plant. In comparison with multi-step chemicalsynthesis, the purification of lutein according to this method is muchmore economical. Furthermore, lutein isolated and purified from marigoldflowers does not contain hazardous chemical impurities as a result ofvarious organic reagents that are normally employed in the sequentialsynthesis of this compound.

The following examples are offered to illustrate but not limit theproduct and process of the present invention.

EXAMPLES Example 1

Saponified marigold oleoresin available commercially as “Kemin YellowOil” was obtained. It was previously processed in the following manner.

Upon receipt the flowers were tested for herbicides and pesticides inorder to ensure that they meet qualifications for food ingredients. Thecompleted oleoresin extract (200 g) was then subjected to saponificationwith aqueous potassium hydroxide. This was accomplished throughcontinuous mixing under heat (65°14 70° C.) of food grade aqueouspotassium hydroxide (45%) and the oleoresin until greater than 98% oflutein was free from fatty acid esters. The saponification was normallycompleted within 35 minutes. The product was then homogenized with amixture of distilled water (700 mL)/ethanol (300 mL, food grade):2.3/1at room temperature for 30 minutes. The mixture was filtered off and thefiltrate was discarded. The retained orange precipitate of lutein waswashed with distilled water until the filtrate was almost colorless andthe pH was neutral. The precipitate was then washed sequentially withcold (0° C.-5° C.) ethanol (200 mL) and hexane (200 mL), respectively.The resulting lutein obtained as orange crystals from three to be 70%pure by spectrophotometric analysis.

Final purification was accomplished by recrystallization from a 1:1mixture of dichloromethane and hexane by dissolving the 70% purecrystals in about 550 ml of dichloromethane containing 1% oftriethylamine. The hexane was added until the solution became cloudy.The cloudy solution was kept at −20° C. to −10° C. to commencerecrystallization. This was completed within about 3 hours resulting inorange crystals of lutein. The crystals were then filtered off andwashed with cold (0° C.) hexane (200 ml) and dried in vacuo at 50° C.for 3 days. The purity of lutein in this instance was greater than 97%.

Example 2

Saponified marigold oleoresin (200 g) was homogenized with a mixture ofdistilled water/ethanol (food grade) at various ratios at 0° C.-10° C.for 30 minutes. The mixture was filtered off and the filtrate wasdiscarded. The retained orange precipitate of lutein was washed withdistilled water until the filtrate was almost colorless and the pH wasneutral. The precipitate was then washed sequentially with cold (0°C.-5° C.) ethanol (200 mL) and hexane (200 mL), respectively. The yieldof lutein obtained as orange crystals from three experiments employingdifferent ratios of water/ethanol are shown below. The lutein obtainedin all three experiments was shown to be about 70% pure byspectrophotometric analysis.

Weight (g) of Crude Lutein Isolated from 200 g Saponi- Ratio ofWater/Ethanol fied Marigold Oleoresin 2.3/1 28 3.0/1 22 1.0/2 15

This example demonstrates the highest yield of lutein is obtained with2.3/1 ratio of water/ethanol. The final purification step byrecrystallization was accomplished as in Example 1, to give lutein inexcess of 97% purity.

The purity and the constituents of the lutein isolated from “KeminYellow Oil” have been determined by UV/visible spectrophotometry as wellas high performance liquid chromatography-photodiode arraydetection/mass spectrometry (HPLC/MS). In addition proton nuclearmagnetic resonance (NMR) spectrum of the purified lutein has beenobtained in order to determine the presence of trace amounts of residualsolvents and other impurities.

UV/Visible Spectrophotometric Analysis. The purity of lutein is greaterthan 97% as determined from the absorption spectrum of this compound inethanol, which exhibits maxima at 422, 446, and 476 nm and extinctioncoefficient of ^(1%) E=2550 in ethanol at 446 nm.

HPLC/MS Analysis: The purified lutein has been examined by HPLC equippedwith a photodiode array detector on both a C₁₈-reversed phase column anda silica-based nitrile bonded column. The HPLC system was interfacedwith a Hewlett-Packard particle beam mass spectrometer. The analysis ofthe purified lutein with HPLC/MS system in addition to HPLC peak puritydetermination at various wavelengths also provided further evidence forpurity of this compound from the total ion chromatogram obtained by massspectrometry analysis.

The HPLC profile of the purified lutein on a C₁₈-reversed phase columnwas observed. 1). As revealed in chromatograms, the purified lutein alsocontains four other carotenoids as minor impurities. In the order ofchromatographic elution on a C₁₈-reversed phase column, the carotenoidsin the purified and recrystallized lutein are: lutein+zeaxanthin(coeluting HPLC peaks, 99.31%), 2′,3′-anhydrolutein (0.23%),α-cryptoxanthin (0.34%), and β-cryptoxanthin (0.10%). Since lutein andits geometrical isomers as well as zeaxanthin (present as a minorcomponent) are not separated on the C₁₈-reversed phase column, a nitrilebonded column was employed. The HPLC profile of the purified lutein onthe nitrile bonded column revealed that under these chromatographicconditions, 2′,3′-anhydrolutein, α-cryptoxanthin, and β-cryptoxanthinappear as one HPLC peak, while lutein and zeaxanthin and several oftheir geometrical isomers are well separated. Based on Quantitative HPLCanalysis of purified lutein on C₁₈-reversed phase and nitrile bondedcolumns, the chemical composition of this compound isolated and purifiedfrom extracts of marigold flowers are shown in Table I.

TABLE I Carotenoid Composition of Lutein Isolated and Purified fromExtracts of Marigold Flowers (Tagetes erecta, variety orangeade)Carotenoids Composition (%) all-E Lutein 94.79 9Z-Lutein 0.14 9′Z-Lutein0.15 13 + 13′Z-Lutein 0.29 Poly Z-Lutein 2.45 Total of Lutein +Z-Isomers 97.82 2′,3′-Anhydrolutein 0.23 All-E-Zeaxanthin 1.51α-Cryptoxanthin 0.34 β-Cryptoxanthin 0.10 Total of Other Carotenoids2.18

From these data it appears that the purified lutein from marigoldflowers consists of 94.79% of all E-lutein, 3.03% of its geometricalisomers (Z-luteins), and a total of 2.18% of 2′, 3′-anhydrolutein,zeaxanthin, α-Cryptoxanthin, and β-Cryptoxanthin. The presence of thelow levels of these carotenoids in the purified lutein product shouldnot be of any concern since these carotenoids are of dietary origin andthey are found routinely at much higher concentrations relative to thatof lutein in human serum/plasma. Individual carotenoids separated by thetwo HPLC columns described above have also been characterized fromcomparison of their absorption and mass spectra determined by aphotodiode array detector interfaced into a mass spectrometer with thoseof standards characterized previously.

NMR Analysis: The proton NMR spectrum of the purified lutein was inagreement with the spectrum of this compound reported previously and noresidual solvents (i.e., dichloromethane, hexane, methanol,triethylamine) or other non-carotenoid impurities (plant sterols andfatty acids) could be detected in this sample.

No substantially pure form of lutein derived from plant extract havingthese levels of purity has heretofore been available.

Example 3 Preparation of Lutein Dose For Oral Supplementation

Carotenoids are considered among the fat soluble nutrients and areusually associated with the liproprotein fractions of human blood.Therefore, the absorption and bioavailability of carotenoids issignificantly increased if these compounds are orally ingested with asmall amount of an oil or foods that contain certain amounts of lipids.In a human study that was conducted the following preparation of luteinresulted in excellent absorption of this compound by the subjects asdetermined from the analysis of their plasma carotenoid profile. In thefollowing, the procedure employed for the preparation of a small batch(100 dose, each 10 mg) of purified lutein is described.

Purified lutein from marigold flowers extract (1 g) was added toabsolute alcohol (75 mL). To this solution α-tocopherol (50 mg) and foodgrade polysorbate 80 (an emulsifier, 4 g) was added and the mixture wassonicated for 10 minutes. The addition of α-tocopherol (0.01% by weight)was to stabilize lutein and prevent this compound from possibleoxidation during long term storage. The above suspension was then mixedwith 350 g of light and mild olive oil (saturated fat/polyunsaturatedfat=2/1) and the mixture was sonicated for 5 minutes. This resulted in asuspension of lutein in olive oil which was stored under nitrogen in arefrigerator. 7 mL aliquots of this suspension was shown byspectrophotometric analysis to contain 10 mg of lutein. At variousintervals, the stability and the purity of lutein suspension in theolive oil was determined by spectrophotometric and HPLC analysis. Thestability studies revealed that lutein prepared and stored under theconditions described above is stable for up to one month. In one studyeach 7 mL aliquot of the olive oil (containing 10 mg lutein) was spreadon a bagel prior to ingestion. Alternatively, this small volume of oilcan be readily taken with other foods. Although this preparation oflutein dose is appropriate for conducting human studies with a limitednumber of subjects, other preparations of this compound into tablets orcapsules can be accomplished. The formulation of β-carotene into tabletsand capsules which has been developed by Hoffman-La Roche Inc. (Nutley,N.J.), may also be found suitable for lutein with some minormodifications.

From the above examples it can be seen that applicant has prepared thefirst ever substantially pure lutein as derived initially from plantextracts. The product is substantially free of impurities, substantiallyfree of other carotenoids, and contains only very low levels of certaincarotenoids of dietary origin. The presence of these carotenoids are ofno concern since they are found routinely at much higher concentrationsin human serum/plasma relative to that of lutein.

It can therefore be seen that the invention accomplishes at least all ofits stated objectives.

1. The carotenoid composition consisting essentially of substantiallypure lutein crystals derived from plant extracts that contain lutein,said lutein crystals being of the formula:

wherein the lutein is substantially free from other carotenoids andchemical impurities found in the natural form of lutein in the plantextract.
 2. The lutein carotenoid composition of claim 1 wherein theplant extract is derived from naturally occurring plants selected fromthe group consisting of fruits, vegetables and marigolds.
 3. The luteincarotenoid composition of claim 1 admixed with an edible triglycerideoil to provide a dose form.
 4. The lutein carotenoid composition ofclaim 1 wherein the lutein is derived from marigold flower extract.
 5. Amethod of obtaining substantially pure, free form lutein crystals fromsaponified marigold extract, comprising the steps of: (a) mixing themarigold extract with water/alcohol mixture remove soluble impurities;(b) lowering the temperature of the mixture to a temperature toprecipitate lutein crystals; (c) washing the lutein crystals with waterto remove water soluble impurities; (d) washing the lutein crystals withan organic solvent mixture to remove organic impurities, and thereafterrecrystallizing the lutein.
 6. The process of claim 5 wherein therecrystallization involves dissolving the lutein crystals in a binarysolvent system and thereafter lowering the temperature to recrystallizelutein in substantially pure form free from other carotenoids andchemical impurities.
 7. The process of claim 6 wherein the binarysolvent system is a halogenated organic solvent in which lutein isstrongly soluble followed by addition of a second organic solvent inwhich lutein is only partially soluble.
 8. The method of claim 7 whereinthe halogenated solvent is dichloromethane, chloroform, or 1,2-dichloroethane.
 9. The method of claim 8 wherein the halogenatedsolvent contains a small but effective amount of an organic base toneutralize trace amounts of acids that are normally present inhalogenated solvents.
 10. The process of claim 9 wherein the organicbase is triethylamine.
 11. The process of claim 10 wherein triethylamineis present at about 1% by volume of the added halogenated solvent. 12.The process of claim 7 wherein the organic solvent is a straight chainhydrocarbon.
 13. The method of claim 5 wherein the ratio of water toalcohol is 2.3 to 1 in step (a).
 14. The method of claim 5 wherein thealcohol in step (a) is selected from the group consisting of ethanol,methanol and isopropyl alcohol.
 15. The method of claim 6 wherein thetemperature is lowered within the range of from about 5° C. to about 10°C.
 16. The method of claim 5 wherein the organic solvent mixture in step(d) includes a straight chain hydrocarbon solvent and an alcohol. 17.The method of claim 16 wherein the temperature of the alcohol and theorganic solvent is from about 0° C. to about 5° C.
 18. The method ofclaim 7 wherein the ratio of the halogenated organic solvent and thesecond organic solvent is 1:1.
 19. The method of claim 6 wherein thetemperature of the solution is lowered to within the range of from −20°C. to −10° C. for from 2 to about 3 hours.
 20. The method of claim 6wherein the recrystallized lutein is dried in vacuo at 50° C. for 3days.
 21. A lutein composition suitable for human consumption whichcomprises: (a) at least about 90 % lutein having been extracted andpurified from plant extracts which contain 10 % or less of non-luteincarotenoids, (b) no traces of toxic chemicals that would render thelutein composition unsuitable for human consumption, and (c)significantly less than about 10 % of non-lutein carotenoids obtained bypurification of said plant extracts.